EBV-EBNA1 Constructs An Immunosuppressive Microenvironment For Nasopharyngeal Carcinoma By Promoting The Chemoattraction Of Treg Cells

Background and Purpose:Nasopharyngeal carcinoma(NPC)is primarily caused by the Epstein-Barr virus(EBV)infection in NPC endemic areas.EBNA1 is an EBV-encoded nuclear antigen,which plays a critical role in the maintenance and replication of EBV genome.However,the mechanisms of EBNA1-promoted NPC immune escape are unknown.Regulatory T(Treg)cells are among the key regulators in restraining antitumour responses.However,the mechanisms of accumulation of Treg cells in NPC have not been defined.This study attempted to identify the detailed mechanisms of EBNA1 functions as a tumour accelerator to promote NPC immune escape by enhancing chemoattraction of Treg cells.Methods:mRNA profiles were determined by next-generation sequencing in NPC cells.In vitro and in vivo assays were performed to analyse the role of EBNA1 in regulation of recruitment of Treg cells.Colocation and coimmunoprecipitation analyses were used to identify the SMAD3/c-JUN complex.Chromatin immunoprecipitation assay and dual luciferase reporter assays were designed to demonstrate c-JUN binding to miR-200a promoter and miR-200a targeting to CXCL12 3'UTR.The hepatocellular carcinoma models were designed to demonstrate universality of the CXCL12-CXCR4-Treg axis in promoting immune evasion of various tumours.Results:1/EBNA1 expression level is positively corelated with Treg cells infiltration degree,patients with EBNA1highTreghigh expression pattern indicates poor prognosis.2/EBV-EBNA1 promotes chemotactic migration of Treg cells by activating TGF?1-SMAD3 signalling.3/Stimulation of the EBV-EBNA1-TGF?1-SMAD3 axis promotes chemotactic migration of Treg cells by reducing miR-200a expression.4/miR-200a suppresses chemotactic migration of Treg cells by directly targeting the CXCL12 3'UTR and CXCL12 suppresses miR-200a transcription.CXCL12 and miR-200a induces a CXCL12-miR-200a-CXCL12 negative feedback loop.5/c-JUN directly binds to miR-200a promoter and induces a c-JUN-miR-200a-CXCL 12-PI3K-AKT-c-JUN feedback loop,which mediates EBV-EBNA1-dependent regulation of chemotactic migration of Treg cells.6/TGF?1 suppresses miR-200a by enhancing the formation of SMAD3/c-JUN protein-protein complex,and EBV-EBNA1 acts as a comprehensive regulator of cellular gene expression by governing the SMAD3/c-JUN interaction in a TGF?1-dependent manner.7/The stimulatory effect of TG?1 on CXCL12 expression is predominantly mediated by the suppression of miR-200a,and chemotactic migration of Treg cells is controlled by the TGF?1-SMAD3-miR-200a-CXCL12 pathway in NPC.8/CXCL12 mediates chemoattraction of Treg cells by upregulating the CXCR4 receptor of Treg cells and the CXCL12-CXCR4-Treg regulatory pathway is universal in various malignant solid tumours.ConclusionsEBV-EBNA1 functions as a tumor accelerator to promote NPC immune evasion.A novel molecular mechanism was identified that involves EBV-EBNA1-stimulated chemotactic migration of Treg cells towards NPC microenvironment by upregulation of the TGF?1-SMAD3-PI3K-AKT-c-JUN-CXCL12-CXCR4 axis and downregulation of miR-200a.Enhanced CXCL12 efficiently attracts CXCR4-positive Treg cells to remodel an immunosuppressive microenvironment.Innovation:We prove a virus-tumor-immunity trinity which reshapes tumor microenvironment and augments tumor immune escape in NPC.